PKR and PKR-like Endoplasmic Reticulum Kinase Induce the Proteasome-dependent Degradation of Cyclin D1 via a Mechanism Requiring Eukaryotic Initiation Factor 2α Phosphorylation

Abstract

Cyclin D1 plays a critical role in controlling the G 1 /S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2␣ (eIF2␣) kinases. We examined the effect of activation of the eIF2␣ kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2␣ phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3␤ and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2␣ phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2␣ phosphorylation during short, but not prolonged, periods of stress.

Alternative ferritin mRNA translation via internal initiation

“…The addition of coumermycin activated GyrB.PKR and resulted in phosphorylation of eIF2a at Ser-51 (Fig. 1A), in agreement with previous observations (Kazemi et al 2004;Raven et al 2008). As expected, this response promoted a shut-off in protein synthesis, as judged by the disappearance of actively translating heavy polysomes in a sucrose gradient fractionation assay (Fig.…”

“…The replacement of the regulatory domain of PKR by the first 220 amino acids of the E. coli GyrB protein, allows dimerization and catalytic activation of the fusion protein upon addition of coumermycin (Ung et al 2001). We utilized human HT1080 fibrosarcoma cells expressing coumermycin-inducible GyrB.PKR (Kazemi et al 2004;Raven et al 2008) as an established model to study ferritin mRNA translation under stress conditions. The addition of coumermycin activated GyrB.PKR and resulted in phosphorylation of eIF2a at Ser-51 (Fig.…”

Expression Profiling and Biochemical Analysis Suggest Stress Response as a Potential Mechanism Inhibiting Proliferation of Polyamine-depleted Cells

“…Cell Cycle Inhibition by Polyamine Depletion-Growth arrest can be manifested as a result of reduced availability of growthstimulating proteins such as cyclin D1, whose level was previously demonstrated to decline rapidly in stressed cells due to attenuated translation and accelerated degradation (40,51,52). We demonstrate here that the level of the cyclin D1 protein declines in DFMO-treated cells (Fig.…”

Endoplasmic reticulum stress: a novel mechanism and therapeutic target for cardiovascular diseases

“…PERK promotes phosphorylation of eIF2α as an immediate response [31] . Increasing unfolded proteins accumulating in the ER induces dissociation of the GRP78/PERK complex and activation of the kinase domain via autophosphorylation [32] , leading to eIF2α phosphorylation and activation that decrease mRNA translation, so proteins gather in the ER [33] . However, the translation of some specific proteins, which include internal ribosomal entry sites such as ATF4, GRP94, and GRP78, is increased via PERK activation [34] .…”